Kinetic studies show that most small proteins unfold and refold in at least two distinct steps, with the slower step having a relaxation time of about one minute at 25 degrees and about one hour at 0 degrees C. Althouh it had been generally accepted that the multiphasic kinetics proved the existence of "structural intermediates" which occur in high concentrations in the transition region, the present work has led to an alternative suggestion which attributes the slowest phase to peptide isomerism of proline residues in the denatured form. Recent studies on proline-containing and proline-free forms of carp parvalbumin provide very strong support of the isomerisms model. In future work, we will first of all attempt to directly "see" the process of proline isomerism occurring during the slow unfolding phase by examining the time dependence of fmr signals of protein which is heavily labelled with 4-fluoro proline. The protein dihydrofolate reductase, has six prolines and will be biosynthesized from proline auxotrophs of Streptococcal faecium, which have been selected for resistance to amethopterin. Second, there is good reason to believe that proline isomerism could cause an intolerable bottleneck in the process of protein synthesis and folding for certain psychrophillic bacteria unless they have a "proline isomerase" present for speeding up the folding of newly-synthesized proteins. The identification and purification of this catalyst will be attempted from strains of psychropillic pseudomonads, using trifluoroacetyl proline and fmr methods to detect catalytic activity in extracts. Once isolated, the isomerase will be used not only to demonstrate the occurrence of proline isomerism in folding reactions but also as a tool for identifying certain native proteins where and equilibrium between two well-folded states may be controlled by proline isomerism; a situation already suggested to occur for concanavalin A. Finally, model compounds will be examined by nmr methods to characterize the occurrence of cis-trans isomerism for non-proline residue. In collaborative efforts with other laboratories, scanning calorimetry will be used to study the structural transitions of myosin systems and also 5s-RNA.